The absorption of dietary phosphate by the small intestine is stimulated by vitamin D3. This stimulation is due primarily to an increase in the amount of the sodium-dependent phosphate transporter Na/Pi-IIb in the brush-border membrane of intestinal enterocytes. In rats, vitamin D3 treatment results in an increase in Na/Pi-IIb mRNA in 2-week-old pups, but not in adults. We plan to define the ontogeny of vitamin D3 stimulation of Na/Pi-IIb expression in rats by studying the effect of Vitamin D3 on the endogenous expression of the Na/Pi-IIb contransporter in rats of different ages and by studying the endogenous expression of the Na/Pi-IIb contansporter in cultured rat intestinal epithelial (RIE-l) cells. The stimulation of vitamin D3 on endogenously expressed Na/Pi-IIb mRNA, protein, and activity in RIE-l cells will be studied in parallel with the measurements of endogenous expression in rats. RIE-l cells transfected with a reporter plasmid in which a luciferase gene is under the transcriptional regulation of the -1103/+15 fragment of the human Na/Pi-IIb 5?flanking region show a significant increase in luciferase expression when exposed to vitamin D3. We will determine the mechanism by which vitamin D3 stimulates human Na/Pi-IIb transcription. We will map the vitamin D3 responsive element(s) in the human Na/Pi-IIb promoter in vitro. RIE-1 cells will be transiently transfected with reporter plasmids which carry a luciferase gene under the transcriptional control of various derivatives of the human Na/Pi-IIb promoter. Truncation mutations and point mutations will be used to identify the location of vitamin D3 responsive elements in the promoter. We will also confirm the physiological significance of the identified response elements in vivo through the use of transgenic mouse colonies which carry constructs with a luciferase reporter under the transcriptional regulation of significant Na/Pi-IIb promoter derivatives identified in the in vitro studies.